WO2007144200A1 - Procédé de liaison covalente de deux molécules par réaction de diels-alder avec demande inverse d'électrons - Google Patents

Procédé de liaison covalente de deux molécules par réaction de diels-alder avec demande inverse d'électrons Download PDF

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WO2007144200A1
WO2007144200A1 PCT/EP2007/005361 EP2007005361W WO2007144200A1 WO 2007144200 A1 WO2007144200 A1 WO 2007144200A1 EP 2007005361 W EP2007005361 W EP 2007005361W WO 2007144200 A1 WO2007144200 A1 WO 2007144200A1
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tetrazine
diene
darinv
dienophile
reaction
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PCT/EP2007/005361
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German (de)
English (en)
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Manfred Wiessler
Eduard Müller
Peter Lorenz
Christian Kliem
Heinz Fleischhacker
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Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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Priority to EP07726066A priority Critical patent/EP2044035A1/fr
Priority to JP2009514713A priority patent/JP5641735B2/ja
Priority to US12/304,982 priority patent/US8552183B2/en
Publication of WO2007144200A1 publication Critical patent/WO2007144200A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/22—Bridged ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/08—Bridged systems

Definitions

  • the present invention relates to a process for covalently linking two molecules using the inverse electron demand Diels-Alder reaction.
  • DAR Diels Alder reaction
  • the Diels Alder reaction is reversible, especially at higher temperatures, and this reversibility is particularly pronounced in the furan / maleimide system, which is due to the high reactivity of the maleinimides for nucleophilic additions
  • the object of the present invention is therefore to provide a method with which complex compounds can be covalently and irreversibly linked to each other and which can also be used for the construction of substance libraries.
  • the object is achieved by a Diels-Alder reaction with inverse electron demand, which comprises the following steps: reaction of a
  • R H, alkyl, aryl, heterocycle, which in turn may be optionally substituted with alkyl, OH, SH, halogen, aryl, heterocycle, nitro, carboxyamido, or amine Group.
  • DARinv inverse electron demand DAR
  • the aim in the synthesis of suitable dienes for the functionalization of peptides, oligonucleotides, surfaces or therapeutics is the preparation of symmetrically or asymmetrically substituted 1, 2,4,5-tetrazines, 1, 2,4-triazines and 1, 2-diazines, which can be easily incorporated into the said bio-molecules.
  • the already known 3,6-dicarboxylic acid ester of 1,2,4,5-tetrazine is not well suited as starting material for the preparation of suitably functionalized 1,2,4,5-tetrazines as dienes.
  • this compound which forms beautiful red crystals, does not have sufficient stability in alcoholic and aqueous solvents; on the other hand, nucleophilic substitution reactions on the ester groups are not possible, since under these conditions the attack of the nucleophile takes place on the tetrazine ring itself. (Kampchen T. et al 1982, Chem. Ber., 115, 683-694.) In the three-step synthesis of this tetrazine, starting with Diazoessigester but the stage of dihydro-1, 2,4,5-tetrazine dicarboxylic acid ester is run through.
  • nucleophilic substitution reactions due to the high reactivity of the ester groups, can easily be carried out on this compound. Since the second nucleophilic substitution, especially with secondary amines, proceeds more slowly than the first, it is also possible to produce monoamides. By suitable reaction, the second nucleophilic substitution can be pushed back, so that dihydrotetrazine monoamides, such as the benzylamide, can be obtained purely by simple recrystallization. This is also an important step in the preparation of functionalized dihydrotetrazine diamides for the derivatization of peptides. It may be advantageous to first introduce the much more stable dihydro-tetrazines as later diene components. After completion of all reactions to be carried out then the oxidation to tetrazine with the immediately following DARinv. Further possibilities for the preparation of more stable monofunctionalized tetrazines based on diaryltetrazines are shown below.
  • a synthetic route for the preparation of triazines consists in the reaction of 1, 2-diketo derivatives with amiddrazones.
  • the tricarboxylic acid ester of 1, 2,4-triazine can be prepared in large quantities according to literature.
  • the ester functions can be easily mixed with nucleophiles, e.g. Convert amines. So far, it has not been possible to discriminate between the ester functions in the reaction with amines.
  • an ester function is sufficient, but sufficient electronegative substituents should then be present to maintain the diene activity of the triazine.
  • Triazines-1, 2,4 are generally less reactive in the DARinv than the tetrazines, but their rate of reaction is still sufficient for ligation reactions, especially when reacted with a very reactive dienophile.
  • the 1,2-diazines which are formed by oxidation of the dihydropyridazines formed in the DARinv of tetrazines with olefins, have even lower DARinv activity than dienes. They are formed directly by the DARinv of tetrazines with triple bonds or with enamines. This also offers the possibility in a sequence, starting from a tetrazine on the diazine by two consecutive DARinv reactions, interrupted by the oxidation of the dihydropyridazine to pyridazine, any two molecules containing a dienophilic anchor group in a predetermined manner to link together ,
  • Tetrazines, triazines and diazines which may be monosubstituted or polysubstituted with electron-withdrawing functional groups are suitable according to the invention as the diene component.
  • electron-withdrawing functional groups may be selected from:
  • the functional groups R which preferably provide functionality for attachment to further molecules (eg to peptides, saccharides or nucleic acids), may be selected from H, alkyl, aryl or heterocycle, where R may in turn optionally be substituted by alkyl, OH, SH, halogen, aryl, heterocycle, nitro, carboxyamido, or amine group.
  • the said functional groups may also be linked directly to the tetrazine, triazine or diazine.
  • Alkyl means Ci - C 2 o, preferably methyl, ethyl, iso-propyl, tert-butyl, etc.
  • the aryl or heterocycle substituents may be selected from: phenyl, - Thienyl, thiophenyl, furyl, furanyl, cyclopentadienyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiazolyl, oxazolyl, Indolyl, furazannyl, pyrrolinyl, imidazolinyl, pyrazolinyl, thiazolinyl, triazolyl, tetrazolyl, and the positional isomers of one or more heteroatoms which may comprise these groups, a group consisting of carbocyclic condensed
  • the diene component may, however, also carry amino acid, peptide, saccharide, lipid or oligonucleotide or nucleic acid substituents at one or more positions.
  • the diene component can also be coupled to all types of pharmaceutical actives, labels, dyes, complexes (e.g., carborane, ferrocene), quantum dots, chelating agents, diagnostics or therapeutics, and combinations thereof
  • Preferred diene components are all esters of the illustrated tetrazines, triazines and diazines and the compounds derived therefrom: e.g.
  • tetrazine monoamides e.g.
  • tetrazine diamides tetrazine-3-trifluoromethyl-6-carboxamides
  • triazine tricarboxylic acid mono-, di- and triamides triazine tricarboxylic acid mono-, di- and triamides
  • 3-carboxyamide-5,6-bis-trifluoromethyl-triazine 1, 2, 4, 1, 2-diazines 3,6, diaryl 4,5-dicarboxylic acid amides e.g.
  • their homologues with ethyl or propyl group can be used instead of methyl.
  • the mentioned tetrazines are relatively easy to prepare by oxidation from the corresponding dihydro compounds, which are accessible via the dihydro-tetrazine dicarboxylic acid ester, which in turn can be easily prepared in two stages from the commercial Diazoessigester
  • a carbocyclic ring is understood as meaning any mono-, bi- or tricyclic carbon ring. These rings may also contain heteratoms. (N, O, S, Si)
  • the dienophile component is also an isolated olefinic double bond or triple bond in a linear or branched hydrocarbon chain, which may optionally contain heteroatoms (N; O, S, Si) in question.
  • both the norbornene anhydrides (exo and endo) and the bicyclic COT anhydride can be readily derivatized with amines, they can be readily converted to functional ligation-suitable molecules.
  • the metathesis reaction also generates ring systems with double bonds.
  • Cyclobutenes can also be generated as reactive dienophiles by photochemical cyclization reactions of cyclic 1,3-dienes, so that the DARinv-based ligation technique can be linked to photolithography.
  • Preferred dienophile components are acids and anhydrides, the functionalized imides and amides derived therefrom and their reduction products, as well as the associated esters and their substitution and reduction products containing a taut or terminal double bond, e.g. exo- or endo-norbornenedicarboxylic anhydride, the two norbornene monocarboxylic esters, cyclobutene monocarboxylic acid esters
  • Cyclohexenedicarboxylic anhydride Sym. Cycloheptencarbonklare, the easily accessible tricyclic COT-MSA adduct, or the corresponding monocarboxylic acid from COT and acrylic acid.
  • the readily accessible 2-allyl-2-propargyl malonate also has two different reactive dienophile groups.
  • Other preferred dienophile components are dehydroproline, allyl-proline, allylmalonic esters, allylgalactose, allyl-silsesquioxane, and all compounds bearing an allyl, butenyl or pentenyl group.
  • the dienophile component may optionally also be monosubstituted or polysubstituted with functional groups.
  • the functional groups may be selected from, for example, alkyl chains (C 2 - C 2 o, methyl, ethyl, iso-propyl, tert-butyl, etc. preferably, optionally halogen-substituted), OH, SH, halogens, aryl, carboxyl , Carbonyl, nitro, carboxyamido, keto, sulfoxide, sulfone, sulfonic acid, sulfide, sulfate, phosphoric acid or amino groups which are bonded directly or via alkyl radicals.
  • the dienophile component may also contain aromatic or heterocyclic radicals.
  • the dienophile component can also carry on one side amino acid, peptide, saccharide, lipid or oligonucleotide or nucleic acid substituents.
  • the dienophile component can also be coupled to all types of pharmaceutical actives, labels, dyes, complexes (e.g., carborane, ferrocene), quantum dots, chelated compexators, diagnostics or therapeutics.
  • Tetrazines have a high reactivity as dienes in the inverse DAR.
  • the model compound for many studies is the readily available tetrazinodicarboxylic acid dimethyl ester.
  • changes to this compound such as nucleophilic substitutions, but lead to decomposition.
  • many reactions of the ester function with nucleophiles can be easily and quickly performed on the dihydro precursor. Since the rate of the first nucleophilic substitution with amines is usually greater than the rate of the second nucleophilic substitution, monosubstituted amides are easily prepared, thus opening the way to the preparation of unsymmetrical diamides.
  • the subsequent oxidation of the dihydro compounds to the tetrazines can be carried out with a variety of oxidizing agents, such as Fe (III) Cl 3 , nitrite, bromine or H 2 O 2 .
  • oxidizing agents such as Fe (III) Cl 3 , nitrite, bromine or H 2 O 2 .
  • a selection of the compounds shown are shown below and illustrate the possibilities of the synthesis concept. This thus makes it possible for every application to produce the analogous connections. It can the Oxidation to tetrazine are carried out at the very end of a reaction sequence because tetrazine dicarboxylic acid amides are much more stable than the Tetrazindicarbonklareester itself.
  • tetrazines with ester functions many tetrazines with aromatic residues are known in the art. They have compared to the tetrazine-dicarboxylic acid dimethyl ester via a reduced diene activity with a significant increase in stability. The greater stability also allows reactions with nucleophiles without destroying the tetrazine ring. Methods are available for preparing unsymmetrically substituted compounds. The introduction of electron-withdrawing substituents such as fluorine, trifluoromethyl or heteroatoms in the phenyl rings leads to an increase in the diene activity. Here, the use of pyrimidine residues has proven to be extremely successful.
  • the monocarboxylic acid amides derived therefrom achieve the diene reactivity of the tetrazine-3,6-dicarboxylic acid ester.
  • the removal of only one nitrogen atom leads to a significant loss of diene reactivity, so that with this series of diaryl monocarboxylic acids a speed range of at least a factor of 10,000 is covered.
  • the goal is the simple representation of monofunctional compounds for incorporation into peptides, oligonucleotides or their anchoring to surfaces.
  • the reactivity as a diene is markedly less pronounced for the triazines.
  • the chemical stability is clear better and is of the order of normal organic compounds.
  • the triazine tricarboxylic acid triethyl ester has been found to be a model compound.
  • Disadvantage of the triazines is in addition to the lower reactivity, the formation of isomeric products in the DARinv.
  • nucleophilic substitution on the ester groups of the triazines by amines is just as feasible as in the tetrazines.
  • due to the lower differences in reactivity unambiguous mono-substitution is not so easily possible. This can be achieved by the use of tert-butyl esters in the building blocks, resulting in the triazine two ester groups of different reactivity.
  • the goal is to introduce nucleophilic substitution with amines functional groups such as carboxy, hydroxy and amino.
  • reporter molecules, therapeutics, peptides or oligonucleotides can then be introduced via these functions.
  • a preferred triazine is also the first-represented 3-carboxymethyl-5,6-bis-trifluoromethyl-triazine 1, 2,4.
  • This triazine has only one function, through which a number of reactive groups can be introduced, which are suitable for the intended use. Through the two adjacent ones Trifluoromethyl groups tend these triazines for hydrate formation, a property that is known by many triazines. This hydrate formation may decrease the activity as a diene in the DARinv, but the DARinv takes place.
  • the DARinv of terazines with a dienophile gives rise to dihydrodiazines, from which diazines are easily accessible by oxidation. If the dienophile is an enamine or a substituted acetylene, the diazines are formed directly. Representation of dienophiles
  • a number of known cyclic and bicyclic unsaturated anhydrides of different ring size can be easily reacted with substituted amines to the corresponding acid imides.
  • Monocarboxylic acids such as, for example, the cyclopentenecarboxylic acid, can also be attached via their acid chlorides to amino acids or diamine ligands.
  • the derivatives of allyacetic acid can likewise be prepared in the same way. These can then be covalently linked by the DARinv to a tetrazine-bearing molecule, even on a surface.
  • the Boc or Fmoc-lysine reactions yield peptide building blocks that can be targeted to any position in peptides. If dienophiles of different reactivity are incorporated into the same peptide chain, these peptides can be marked several times in a targeted manner. The same applies to oligonucleotides
  • a special case is that of cyclooctatetraene, COT for short, and MSA readily accessible tricyclic anhydride.
  • the DAR of the bicyclic form of the COT results in a molecule containing two different reactive dienophiles for the DARinv.
  • Other functions can be introduced in the usual way via the anhydride ring, such as amino acids, amines or the coupling to a solid phase.
  • the following amino acids can be purchased and all dr ⁇ fi react as dienophiles in the DARinv.
  • the three amino acids can be introduced at any position during the peptide synthesis and then new residues can be introduced via the DARinv at these positions; Such peptides can also be anchored to surfaces in a defined manner for detailed structural investigations.
  • the enzymatic peptide synthesis also allows the introduction of amino acids with dienophilic anchor groups as the group of Bertozzi has shown.
  • the building blocks for the peptide synthesis represented by the inventors are summarized above.
  • the reverse principle namely the incorporation of the diene into the peptide can also be carried out on the solid phase.
  • a lysine is coupled, which is substituted at the amino group with a tetrazine.
  • the peptide can be isolated as a pink solid.
  • the implementation with the COT-Lys-EILDV Peptide leads to a coupled dipeptide.
  • the same peptide is isolated when the DARinv is carried out immediately on the solid phase.
  • the peptides can also be labeled either on the solid phase or after cleavage by the DARinv with dyes or biotin.
  • the aim is to produce platinum complexes in which either the diamine ligand or the dicarboxylic acids used as the leaving group are formed as dienophile or as diene.
  • the structures listed below are intended to illustrate this concept. This makes it possible to selectively the respective part of the Complex by the DARinv targeted change. This makes possible the construction of libraries of the original complex.
  • the change in the leaving group, in this case the dicarboxylic acid, by the DARinv appears to be particularly interesting since this leaving group is split off during the intracellular activation of the Pt complexes and thus also the respective part attached by the DARinv.
  • the leaving group could be targeted by the DARinv, for example by peptidic signal sequences, or other biomolecules that can mediate preferential uptake of these complexes in tumor cells.
  • the expression of the diamine ligand as a dienophile allows, after covalent binding of the active platinum complex to the DNA to detect the localization of the resulting adduct by a DARinv with a reporter molecule .
  • both the amine ligands and the leaving group as dienophiles with different Dienophile activity, which allows simultaneous targeted ligation of both the amine ligand and the leaving group.
  • the Pt complexes shown here were prepared ,
  • platinum complex of 1,2,3-triaminopropane shown is known, it can be bound to the tetrazine via its free amino group, thus providing another building block for the incorporation of Pt complexes into proteins, saccharides and other biomolecules and therapeutics.
  • Positron emission tomography is a radioactive, non-invasive, but very sensitive diagnostic method.
  • the most commonly used positron emitter is F18, which decays into the element oxygen with a half-life of 18 min, releasing a positron. Because of the low Half life requires the preparation of appropriate F18-labeled compound in particular synthetic methods. They have to run fast and necessary cleaning procedures have to be easy.
  • the most commonly used compound today is 2-fluoro18-2-deoxy-glucose.
  • the ligation reaction based on the DARinv can be used very well for labeling peptides, oligonucleotides and saccharides with F18.
  • Nucleophilic substitution reactions on aromatics are facilitated as the number of nitrogen atoms in the ring increases, such as in the series benzene, pyridine, pyrimidine and triazine.
  • a thiomethyl radical can easily be replaced by a number of nucleophiles, including halogens.
  • triazines react as dienes in the DARinv
  • the prior introduction of F18 into such a triazine offers the elegant possibility to label the abovementioned biopolymers with F18 with the aid of the DARinv and thus make them accessible for detection by PET.
  • the classical way of nucleophilic substitution on tosylates with fluoride can be labeled (see below).
  • the introduction of trifluoroacetyl groups labeled F18 via the amine function of the described tetrazines and triazines can be used here.
  • the tetrazine can be trifluoacetylated in pyridine and directly in the Solution that DARinv perform.
  • the DARinv allows very short reaction times and usually proceeds without the formation of by-products.
  • the dihydro-tetrazine dicarboxylic acid ester can be reacted with amines at RT with primary amines.
  • This high reactivity can be used to build up reactive solid phases.
  • the reaction sequences shown here can be carried out in yields between 70 and 90%.
  • solid phases are available which carry either a diene or a dienophile for the DARinv.
  • the reactivity in the DARinv is so high that the dienophile-bearing solid phase can be literally titrated with a tetrazine.
  • the resulting applications range from the chip technology for oligonucleotides, proteins or saccharides to catalytic surfaces and solid phase reagents.
  • trimethoxy-silyl compounds for anchoring to surfaces, in which case both the diene and the dienophile can be anchored to the surface.
  • acid chlorides of the diaryl-tetrazine monocarboxylic acids and of the dihydro-tetrazine-glycic acid chlorides are also available for this purpose.
  • DARinv Since the DARinv is a very fast reaction, two surfaces can be covalently bonded to each other in the sense of an adhesive. Surface reactions are usually slower than reactions in solution. At DARinv we have the observation made that the driving force of the reaction is so great that even in those cases in which the tetrazine is poorly soluble, such as in water, the reaction takes place at the surface of the tetrazine particles under visible evolution of nitrogen.
  • the analytical methods for the detection of DNA adducts are still not automatable.
  • the P32 post-labeling method is still the most sensitive method, but so far no method is available that allows the simultaneous detection of different adduct types.
  • the method of separation by capillary electrophoresis with subsequent fluorescence detection is very well suited for the detection of 5-methyl-cytosine, but missed the detection limit of the 32P-post.labeling method by at least a factor of 100.
  • the present invention described ligation reaction.
  • the amine derived from norbornene can be coupled to the phosphate group of the nucleosides by a more recent method according to literature.
  • any fluorescent dye linked to a tetrazine or triazine can be coupled via the DARinv and subsequently detected by means of capillary electrophoresis.
  • any substituents can be introduced into oligonucleotides obtained by synthesis.
  • the required amidites have been prepared by and. In this way, a multiple mark is possible.
  • the photochemical cyclization of 1,3-dienes to cyclobutenes proceeds with high quantum yield, generating a highly reactive inverse dienophile.
  • the photolithography can be linked to the ligation by the DARinv. Because only where UV light is irradiated, the 2 + 2 cycloaddition takes place and only there can then proceed the DARinv.
  • substituted tetrazines and triazines can also be added to surfaces here.
  • peptides or saccharides for use as therapeutics, diagnostic agents or for the study of Interacting of peptides or saccharides with each other or with other biomolecules.
  • the dihydro-tetrazinedicarboxylic acid can also be incorporated into polyamides during the condensation and, after oxidation to tetrazine, modified by DARinv.
  • the diaryl-tetrazine-dicarboxylic acids are also suitable for incorporation in nylon / nylon-type polyamides. If tetrazines with different diene reactivity are incorporated in statistical distribution, they can be specifically modified in different ways.
  • Oligomeric tetrazines or mixed oligomeric tetrazines / triazines can be prepared with the above-described building blocks and then selectively modified by DARinv. Again, the corresponding polymers are conceivable. Polymeric tetrazines
  • Quantum dots are nanoparticles made up of compounds such as CdS or CdSe and have special optical properties. Upon excitation with lasers they fluoresce very strongly depending on their size and therefore find more and more use in the diagnostic field, especially as they make the detection of single molecules possible. The prerequisite for this, however, is their doping with functional groups which proceeds via SH groups and permits a subsequent interaction with the molecules to be detected. Due to their special properties, gold nanoparticles are used for electron microscopic investigations of biomolecules. Again, the anchoring of molecules on the surface via SH groups accomplished. Again, the new ligation technique can be used by DARinv.
  • SH-group-containing triazines and tetrazines were prepared, wherein first the disulfides were prepared and from this by reduction with dithiothreitol the mercapto compound.
  • SH-containing dienophiles of the norbornene type can also be prepared. The disulfides themselves were also anchored to gold surfaces.
  • both the dienes tetrazines, triazines and diazines
  • dienophiles can be deposited via the disulfide group as to the surface of the quantum dots or other metals and are therefore accessible to the DARinv.
  • antibodies, peptides, saccharides or therapeutics can be anchored to the surface of the quantum dots for diagnostic or therapeutic purposes.
  • the synthesis of complex saccharide structures requires a sophisticated protecting group strategy.
  • the reducing end is often protected by an allyl group or a pentenoyl group.
  • isolated oligosaccharides can be easily provided with an allyl group or pentenoyl group at the reducing end.
  • the conditions for anchoring these oligosaccharides to a solid phase or surface are given by the DARinv.
  • the furan saccharide mimetics described in DE-A-100 41 221.1 can likewise be anchored either to biomolecules or surfaces with the aid of this DARinv technology.
  • Prerequisite is the introduction of an allyl ether group or the use of linker molecules as described above.
  • saccharide mimetics are also suitable as inverse dienophiles in the DARinv and can thus be introduced into any biomolecules.
  • vitamin A, vitamin C, curcumin or other therapeutic agents can be coupled to proteins or surfaces and released there by hydrolysis or enzymatic cleavage.
  • Another example of this application is the temozolomide used to treat brain tumors, which can be coupled via its acid function either to a tetrazine or to a dienohile.
  • the DARinv then the coupling to peptides is possible.
  • liposomes containing double bonds that are active in the DARinv can be easily modified with this technology, thereby improving targeting or modification
  • Both systems are suitable for the sequential coupling of molecules which carry either the same dienophile as L 1820 or different dienophiles such as L 1825.
  • This type of compound is also suitable for anchoring to a solid phase by reaction with the carbonyl or the carbonyl group
  • the ketone dicyclopentadienone is an orthogonal dienophile that is accessible to both normal DAR and DARinv. By reaction with butadiene, it can also be converted into a double inverse dienophile (s. In addition to these linkers from dienophiles, linkers from dienes that allow DAR and DARinv at the same time are also available.
  • linkers from dienophiles linkers from dienes that allow DAR and DARinv at the same time are also available.
  • the combination of tetrazine or triazine with a dienophile, such as a maleimide, for the classical DAR is possible. The structures are listed below.
  • novel tetrazines, triazines and diazines as dienes in the DARinv can be with dienophiles, such as enamines, enol ethers, etc., build novel libraries of substances to search for new drugs.
  • the diamides themselves are, as described, accessible in large numbers by successive reactions with various amines and subsequent oxidation. This reaction sequence can be automated.
  • sufficient water solubility is an important prerequisite.
  • One possible approach to improving water solubility and thus oral availability is the covalent attachment of drugs to saccharides to form conjugates.
  • Fig. 2 Diels-Alder reaction with inverse electron demand
  • the peptide was synthesized on the synthesizer, with the previously described lysine derivative added with the tricyclic dienophile. This peptide was purified by HPLC and the structure confirmed by the mass spectrum. Molecular peak at m / e 899.
  • Dihydro-tetrazine-3,6-dicarboxylic acid dimethyl ester 200 mg (1 mmol) are suspended in 5 ml of methanol and (2.5 mmol) glycine methyl ester in 5 ml of methanol - from 2.5 mmol glycine methyl ester hydrochloride and 2.5 mmol triethylamine prepared - added dropwise and stirred overnight at room temperature.
  • the reddish solution has turned pale yellow and a yellow precipitate has formed. After cooling to -18 ° C, this precipitate is filtered off and recrystallized from methanol. Yield 55%.
  • Mass spectrum and NMR confirm the structure.
  • the resulting silica gel was suspended in ethyl acetate and shaken with a 5-fold excess of isoamyl nitrite for 5 hrs at room temperature. It was filtered, washed several times with ethyl acetate and ether and dried. The silica had now assumed a light pink color.
  • the elemental analysis revealed only minor changes in the C / N ratio.
  • the Diels Alder reaction was carried out with the already used tricyclic anhydride. 100 mg of the tetrazine-loaded silica gel were suspended in ethyl acetate and shaken with 0.3 mmol of the anhydride for 2 hours at room temperature. It was filtered again, washed 5 times with ethyl acetate and dried.
  • the C / N ratio of the elemental analysis confirmed the original specific silica occupancy with dihydrotetrazine at about 70%.
  • Amino-functionalized silica gel (1g.) was suspended in 10 ml of ethanol, 2 mmol (400mg) of the tricyclic anhydride was added and shaken for 3 hrs. At 80 0 C. It was filtered off with suction through a frit, washed several times with ethanol and finally with ether and dried. The determination of the C / N ratio by elemental analysis revealed a coverage of 70% of the available amino groups.
  • the triazine tri-carboxylic acid methyl ester 1 mmol (255 mg) was dissolved in 2 ml THF and treated dropwise with a solution of 1 mmol (248 mg) of the tricyclic dicarboxylic acid dimethyl ester in 1 ml of THF. It is observed nitrogen evolution and a color lightening. After 2 hours at room temperature, the mixture is concentrated and the residue is chromatographed on silica gel with hexane / ethyl acetate 1: 1. 250 mg of the adduct are isolated corresponding to 50% yield. The mass spectrum confirms the structure, molecular peak at m / e 475.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Indole Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

La présente invention concerne un procédé de liaison de deux molécules par réaction de Diels-Alder avec demande inverse d'électrons, ledit procédé comprenant les étapes consistant à : faire réagir une (a) triazine ou tétrazine avec un ou plusieurs substituants accepteurs d'électrons sur le cycle, en tant que composant diène, les substituants accepteurs d'électrons étant choisis parmi : - COOR - C(O)NR2 - CX3 (X = halogène) - halogène -CN -SO2-R ou SO3-R - PR2 avec R = H, alkyle, aryle, hétérocycle qui peuvent éventuellement à nouveau être substitués par les groupements alkyle, OH, SH, halogène, aryle, hétérocycle, nitro, carboxyamido ou amine, des cycles hétérocycliques avec 1, 2 ou 3 atomes N, O ou S, avec une taille de cycle de 5 ou 6 atomes, substitués par au moins un groupement carboxyle, acide sulfonique ou phosphonique, avec (b) une double ou triple liaison isolée définiques dans un cycle (hétéro)carbocyclique ou une double ou triple liaison isolée dans une chaîne hydrocarbonée linéaire ou ramifiée, pouvant éventuellement contenir des hétéroatomes, en tant que composants diénophiles.
PCT/EP2007/005361 2006-06-16 2007-06-18 Procédé de liaison covalente de deux molécules par réaction de diels-alder avec demande inverse d'électrons WO2007144200A1 (fr)

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JP2009514713A JP5641735B2 (ja) 2006-06-16 2007-06-18 逆電子要求性ディールズ・アルダー反応を伴う２つの分子の共有結合方法
US12/304,982 US8552183B2 (en) 2006-06-16 2007-06-18 Process for the covalent coupling of two molecules by means of a diels-alder reaction with inverse electron requirement

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EP06012414A EP1867638A1 (fr) 2006-06-16 2006-06-16 Procédé pour le lien de deux molécules par une réaction Diels-Alder avec une demande d'électron inverse

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CA3236912A1 (fr) * 2021-11-05 2023-05-11 Georgiamune Inc. Modulateurs d'akt3

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1770098A1 (de) * 1968-03-30 1971-09-23 Basf Ag Verfahren zur Herstellung von Verbindungen der Pyridinreihe
WO1998016508A2 (fr) * 1996-10-02 1998-04-23 Trustees Of Boston University Synthese de composes aromatiques au moyen de la reaction diels-alder sur support solide
EP1243579A1 (fr) * 1999-11-30 2002-09-25 Sankio Chemical Co., Ltd. Procede relatif a l'elaboration de derives de pyridine

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2180758T3 (es) * 1995-05-04 2003-02-16 Aventis Pasteur Vacunas de pertussis acelulares y metodos de preparacion de las mismas.

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1770098A1 (de) * 1968-03-30 1971-09-23 Basf Ag Verfahren zur Herstellung von Verbindungen der Pyridinreihe
WO1998016508A2 (fr) * 1996-10-02 1998-04-23 Trustees Of Boston University Synthese de composes aromatiques au moyen de la reaction diels-alder sur support solide
EP1243579A1 (fr) * 1999-11-30 2002-09-25 Sankio Chemical Co., Ltd. Procede relatif a l'elaboration de derives de pyridine

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ANTONIO M D'A ROCHA GONSALVES ET AL: "DIELS-ALDER REACTIONS OF 1,2,4,-TRIAZINES WITH CYCLIC VINYL ETHERS", TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 49, no. 24, 1993, pages 5277 - 5290, XP002936542, ISSN: 0040-4020 *
KISELEV V D ET AL: "Volume, Enthalpy and Entropy of Activation of the Diels-Alder Reaction of Dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate with 1-Hexene", TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 55, no. 41, 8 October 1999 (1999-10-08), pages 12201 - 12210, XP004178988, ISSN: 0040-4020 *
LIPINSKA T: "Experimental and theoretical FMO interaction studies of the Diels-Alder reaction of 5-acetyl-3-methythio-1,2,4-triazine with cyclic enamines", TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 61, no. 34, 22 August 2005 (2005-08-22), pages 8148 - 8158, XP004985234, ISSN: 0040-4020 *
PANEK J S ET AL: "Synthesis of Aromatic 1,2-Diazines by Inverse Electron Demand Diels-Alder Reaction of Polymer-Supported 1,2,4,5-Tetrazines", TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 37, no. 45, 4 November 1996 (1996-11-04), pages 8151 - 8154, XP004031068, ISSN: 0040-4039 *
See also references of EP2044035A1 *
SPAREY T J ET AL: "Inverse Electron Demand Diels-Alder Reactions of 3,6-Dichloro-[1,2,4,5]tetrazine", TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 39, no. 32, 6 August 1998 (1998-08-06), pages 5873 - 5874, XP004161315, ISSN: 0040-4039 *
STEHL A ET AL: "Racemic and enantiopure 4-(piperidine-2'-yl)-pyridazines: novel synthesis of anabasine-analogues with potential nicotinic acetylcholine receptor agonist activity-a new approach via Diels-Alder reaction with inverse electron demand", TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 58, no. 7, 11 February 2002 (2002-02-11), pages 1343 - 1354, XP004334839, ISSN: 0040-4020 *
TAYLOR E C ET AL: "INTRAMOLECULAR DIELS-ALDER REACTIONS OF 1,2,4-TRIAZINES. SYNTHESIS OF 2,3-CYCLOPENTENOPYRIDINES AND 5,6,7,8-TETRAHYDROQUINOLINES", JOURNAL OF ORGANIC CHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, US, vol. 56, no. 5, May 1991 (1991-05-01), pages 1807 - 1812, XP002921410, ISSN: 0022-3263 *
WAN Z-K ET AL: "Dienophilicity of imidazole in inverse electron demand Diels-Alder reactions: cycloadditions with 1,2,4,5-tetrazines and the structure of zarzissine", TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 57, no. 26, 25 June 2001 (2001-06-25), pages 5497 - 5507, XP004247085, ISSN: 0040-4020 *

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